Comparison of bone marrow extracellular matrices.

We have compared the structure and composition of adult and fetal bovine bone marrow extracellular matrices. In contrast to fetal bone marrow, adult bone marrow has more oval fenestration and accumulation of adipocytes as well as lower protein content. These differences could be due to remodeling of bone marrow tissue as it develops. Zymogram analysis of matrix metalloproteinase (MMP) and tissue inhibitor of MMP (TIMP) activities showed that fetal, but not adult bone marrow extract contained a 96-kDa MMP and TIMP-1 and -2. These activities may contribute to the structural differences between adult and fetal bone marrow tissues.     

Random Amplified Polymorphic DNA Variation among and within Selected Ixora (Rubiaceae) Populations and Mutants

Random amplified polymorphic DNA (RAPD) analysis was used tostudy variation among and within selectedIxora (Rubiaceae) populationsand mutants. Six populations of I. congesta yielded identicalbanding patterns suggesting genetic uniformity of this species.However, six populations of I. coccinea varieties (three red-flowered,two yellow-flowered and one red-flowered wild-type) exhibitedinfraspecific differences in RAPD profiles. Small and largeleaves of an atavistic mutant cultivar of I. coccinea were alsosubjected to RAPD analysis. An extra band was amplified in thelarge leaves that was absent in small leaves, suggesting thatthe phenotypic alteration in this taxon is due to genetic mutationrather than epigenetic changes. Similarly, an extra band wasdetected in the white sectors of I. Variegated compared to thegreen sectors, suggesting that the shoot apical meristems ofthis cultivar exist as a genetic chimera. DNA gel blot hybridizationwas performed to confirm the specificities of selected bands.Our study indicates that differences among individuals of variouspopulations and mutants may be detected using RAPD markers.Copyright 1999 Annals of Botany Company Ixora L., variegated variety, RAPD fingerprinting, DNA gel blot, intraspecific genetic similarity, atavistic mutant.     

Cytochrome P450 2E1-derived reactive oxygen species mediate paracrine stimulation of collagen I protein synthesis by hepatic stellate cells.

To evaluate possible fibrogenic effects of CYP2E1-dependent generation of reactive oxygen species, a model was developed using co-cultures of HepG2 cells, which do (E47 cells) or do not (C34 cells) express cytochrome P450 2E1 (CYP2E1) with stellate cells. There was an increase in intra- and extracellular H(2)O(2), lipid peroxidation, and collagen type I protein in stellate cells co-cultured with E47 cells compared with stellate cells alone or co-cultured with C34 cells. The increase in collagen was prevented by antioxidants and a CYP2E1 inhibitor. CYP3A4 did not mimic the stimulatory effects found with CYP2E1. Collagen mRNA levels remained unchanged, and pulse-chase analysis indicated similar half-lives of collagen I protein between both co-cultures. However, collagen protein synthesis was increased in E47 co-culture. Hepatocytes from pyrazole-treated rats (with high levels of CYP2E1) induced collagen protein in primary stellate cells, and antioxidants and CYP2E1 inhibitors blocked this effect. These results suggest that increased translation of collagen mRNA by CYP2E1-derived reactive oxygen species is responsible for the increase in collagen protein produced by the E47 co-culture. These co-culture models may be useful for understanding the impact of CYP2E1-derived ROS on stellate cell function and activation.     

Crystal structure of a novel mid-gut procarboxypeptidase from the cotton pest Helicoverpa armigera.

The cotton bollworm Helicoverpa armigera (Hubner) (Lepidoptera: Noctuidae) is one of the most serious insect pests in Australia, India and China. The larva causes substantial economical losses to legume, fibre, cereal oilseed and vegetable crops. This pest has proven to be difficult to control by conventional means, mainly due to the development of pesticide resistance. We present here the 2.5 A crystal structure from the novel procarboxypeptidase (PCPAHa) found in the gut extracts from H. armigera larvae, the first one reported for an insect. This metalloprotease is synthesized as a zymogen of 46.6 kDa which, upon in vitro activation with Lys-C endoproteinase, yields a pro-segment of 91 residues and an active carboxypeptidase moiety of 318 residues. Both regions show a three-dimensional structure quite similar to the corresponding structures in mammalian digestive carboxypeptidases, the most relevant structural differences being located in the loops between conserved secondary structure elements, including the primary activation site. This activation site contains the motif (Ala)(5)Lys at the C terminus of the helix connecting the pro- and the carboxypeptidase domains. A remarkable feature of PCPAHa is the occurrence of the same (Ala)(6)Lys near the C terminus of the active enzyme. The presence of Ser255 in PCPAHa instead of Ile and Asp found in the pancreatic A and B forms, respectively, enlarges the S1' specificity pocket and influences the substrate preferences of the enzyme. The C-terminal tail of the leech carboxypeptidase inhibitor has been modelled into the PCPAHa active site to explore the substrate preferences and the enzymatic mechanism of this enzyme.     

Molecular phylogeny of the fungus gnat subfamilies Gnoristinae and Mycomyinae,and their position within Mycetophilidae (Diptera)

The phylogeny of the fungus gnat family Mycetophilidae (Diptera) is reconstructed with a focus on the species‐rich and taxonomically difficult subfamilies Gnoristinae and Mycomyinae. The multigene phylogenetic analyses are based on five nuclear (18S, 28S, CAD, MCS, ITS2) and four mitochondrial (12S, 16S, COI, CytB) gene markers. The analyses strongly support the monophyly of Mycetophilidae and the subfamilies Manotinae, Sciophilinae, Leiinae, and Mycomyinae, although Gnoristinae is paraphyletic with respect to Mycetophilinae. All the genera and groups of genera included are supported as monophyletic, except for Acomoptera Vockeroth, Boletina Staeger, Dziedzickia Johannsen, Ectrepesthoneura Enderlein, and Neoempheria Osten Sacken. Ancestral character state reconstructions were applied to two morphological features present in Gnoristinae and Mycomyinae (i.e. presence of setae on wing membrane and wing vein R₄) in order to assess their evolution. The wing vein R₄ appears as an unstable character, spread throughout different clades. A dated phylogeny of the family Mycetophilidae showed that most of the subfamilies of Mycetophilidae originated and diversified during the Cretaceous. The youngest subfamilies, originated in the Paleogene, appear to be Mycomyinae and Mycetophilinae.     

The conversion of tubulin carboxyl groups to amides has a stabilizing effect on microtubules.

In a recent study, we demonstrated that the conversion of carboxyl residues in the C-termini of tubulin to neutral amides with glycine ethyl ester enhanced the ability of the protein to assemble into microtubules and decreased its interaction with microtubule-associated proteins (MAPs). In this work, we investigated the effects of carboxyl modification on the dynamic behavior of microtubules at polymer mass steady state. After steady state, microtubules assembled from unmodified tubulin were sheared, and the mean polymer lengths decreased to 5 microns and then increased to 29 microns within 130 min. In contrast, lengths of sheared microtubules polymerized from tubulin containing 23 modified carboxyl groups increased by only 2-fold. Stabilization of polymer lengths was also observed directly by video-enhanced light microscopy of microtubules grown off of axonemes. Rapid shortening was seen in microtubules composed of unmodified but not modified tubulin. Further evidence for the less dynamic behavior of microtubules as a result of carboxyl modification was obtained from kinetic studies of the elongation phase during assembly which showed a 3-fold lower off-rate constant, k-, for modified microtubules. Another effect of the modification was a 12-fold reduction in the steady-state rate constant for GTP hydrolysis (165 s-1 for unmodified and 14 s-1 for modified). These results suggest that reduction of the negative charges in the C-termini by modification of the acidic residues stabilizes microtubules against depolymerization. MAPs may stabilize microtubules in an analogous manner.